HS Treatment Research Articles

Effects of supplemental molybdenum on animal performance, liver copper concentrations, ruminal hydrogen sulfide concentrations, and the appearance of sulfur and molybdenum toxicity in steers receiving fiber-based diets1

Poor performance and S-induced polioencephalomalacia (sPEM) have been observed in ruminant livestock in high-S drinking water regions. No gainful method of removing S from drinking water is available and therefore a feed supplement that negates the effects of high-S water is needed. Our objective was to determine if supplementing Mo improves health and performance of steers administered a high-fiber diet and high-S drinking water. We hypothesized that if the supplemental Mo adequately bound excess S in the rumen, it would not be available at toxic concentrations. Yearling steers (n = 96; 260.0 ± 1.3 kg BW) were stratified by pretrial BW into 12 feedlot pens (n = 8 steers per pen). One of 3 treatments, low-S water (LS; 375 mg SO(4)/L), high-S water (HS; 2,218 mg SO(4)/L), or high-S water plus Mo (HSMO; 2,218 mg SO(4)/L; 187.5 mg Mo/kg DM), were randomly assigned to pens within 4 blocks for a 56-d trial. Body weights were recorded on d -2, -1, 29, 56, and 57, ruminal H(2)S concentrations were measured by rumenocentesis on d -1, 29, and 57, and liver biopsies were performed on d -1 and 57. Performance data were analyzed over the 56-d trial period (overall) as well as over 2 periods: Period 1 (d 0 to d 28) and Period 2 (d 29 to d 56). One case of sPEM was confirmed by the presence of cortical lesions in the HS treatment group. Daily DMI and ADG were affected by treatment and period (P < 0.001) main effects. The LS steers had the greatest (P < 0.05) DMI followed by HS and HSMO steers, respectively. Similar results were observed for ADG. Daily water intake was affected (P < 0.001) by period only, with greater daily water intake in Period 2 than Period 1. Change in hepatic concentrations of Cu, Fe, and Mo over the course of the trial were all affected (P < 0.001) by treatment. Hepatic Cu increased from d 1 to 57 in LS and HS steers but was depleted in HSMO steers. Hepatic Fe and Mo increased in HSMO steers only. Ruminal H(2)S concentrations were affected by treatment (P < 0.021), with greater H(2)S concentrations in HSMO compared with LS and HS steers. Signs of Mo toxicity such as severe diarrhea, loss of body condition, anorexia, changes in hair color, and stiffness in joints were observed in the Mo supplemented steers. These results indicate that added dietary Mo does not adequately bind excess S in the rumen, causing aggravated toxic effects from potentially both the high dietary S and Mo.

Effect of Deep Sea Water on Seed Germination, Photoperiod and Temperature on the Growth and Flowering of Buckwheat Species

Experimental Farm, Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, JapanAbstract - This paper describes the potential use of deep sea water to stimulate seed germination in both common and Tartary buckwheat. Treatment of 10% deep sea water at 25℃ would slightly enhance germination of buckwheat seeds compared to non-DSW treatment and other temperature. In this study, the significant effects of photoperiod and temperature on seedling growth were also found in the HL treatment for the number of leaf, plant height, and plant fresh weight and LL treatment for root length and leaf size. Common buckwheat (Suwon No.1) showed higher rate (93%) of flowering plants in the HS and LL (93% of flowering rates) than those revealed in the HS and LS treatment, while the low percentage(67%) of plant flowering plants was shown in the LS treatment. All plants (100%) of a Korean landrace, Ahndong-jaerae showed flowers in the HS and LS treatment. HL and LL treatment status did not occur in the plant's flowering. Any Tartary buckwheat (KW45) plant did not yet flowered when it was 21 days-old.Key words - Deep sea water, Germination, Photoperiod, Temperature, Seedling growth, Flowering*Corresponding author. E-mail : chpark@kangwon.ac.krⓒ 2012 by The Plant Resources Society of Korea

Abstract 5115: Genetic signature of histiocytic sarcoma revealed by an unbiased genetic screen in mice

Abstract Histiocytic Sarcomas (HS) are rare neoplasms derived from hematopoietic or mesenchymal stem cells and are known to have a poor prognosis. Primary lesions of HS appear in the spleen, lymph nodes, lung, bone marrow, skin and hypodermis. The definitive cell of origin, genetic etiology, and critical drug targets for HS therapy are unknown. To address all three questions we have utilized a forward genetic screening method in mice that can identify mutations capable of causing cancer initiation and progression. The forward genetic screen uses the Sleeping Beauty (SB) DNA transposon as a random somatic mutagen, capable of both activating proto-oncogenes and inactivating tumor suppressor genes. The system relies on the use of a Cre/LoxP-regulated SB transposase transgene and mutagenic transposon vector transgenes that are mobilized by the transposase. Using Cre recombinase under the control of a tissue specific promoter allows for selective, tissue specific mutagenesis in particular cells of interest. Tumors generated in these mice are analyzed for recurrent transposon insertion sites (called common insertion sites or CIS) using ligation-mediated PCR in combination with high-throughput sequencing. As in human cancer, our results often reveal a small number of frequently mutated genes that cooperate with a greater number of rare mutant genes that drive formation of tumors. We hypothesized that SB-mediated transposition of an oncogenic transposon in myeloid lineage cells in mice could cause mutations in oncogenes and tumor suppressor genes leading to the initiation and progression of HS. We generated and aged 190 experimental and control transgenic mice. The triple transgenic, experimental mice express SB transposase in myeloid lineage cells containing an oncogenic transposon by virtue of a Cre recombinase transgene under the control of the Lysozyme M promoter, which is expressed in committed myeloid lineage cells. Remarkably, triple transgenic mice became moribund much faster than controls. Histopathology suggests 30% of the experimental mice developed HS. We have performed immunohistochemistry to confirm the tumor type using antibodies to Mac-2, F4/80 and PAX5. Tumors were analyzed by mapping transposon insertions to the mouse genome from many independently generated tumors. We mapped 1,177 non-redundant insertions and generated a list of 23 candidate HS genes comprised of known cancer genes as well as novel candidate genes. Our top 3 hits are Raf1 and Fli1, known oncogenes associated with hematological neoplasms, and Mitf, a transcription factor associated with melanoma. Our list also includes known tumor suppressors Trp53 and Nf1. Our results show that 1: A mature myeloid lineage cell can serve as a target cell for HS. 2: ETS and basic helix-loop-helix (hHLH)-leucine zipper transcription factor networks can induce HS with Raf-activated pathways And 3: RAF-MEK-ERK signaling is likely a useful target for HS treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5115. doi:1538-7445.AM2012-5115

Establishing a New Zebrafish Model to Study Malignant Transformation in Myeloproliferative Disorders

Abstract 4711 Background:Myeloproliferative diseases (MPDs) are a group of hematological malignancies characterized by the abnormal increase of different blood cells in peripheral blood and in other hematological organs. The molecular pathways involved in disease pathogenesis and progression as well as in drug sensitivity and resistance are not fully elucidated yet. Given the physiological similarity to mammals, the zebrafish (ZF) could accelerate the study of the molecular basis of these diseases. In the last decade several human leukaemia associated oncogenes have been transiently over-expressed in ZF embryos in order to perturb the myelo-erythroid compartment, with the aim to create experimental models to discover new molecular pathways that can became potential targets for new chemical compounds. Methodogy:We took advantage of the binary Gal4/UAS system to express oncogenic human HRAS in the ZF hematopoietic compartment. We used the tg(MAZe) driver line, a specific transgenic line that allows random mosaic expression of Gal4-VP16 after heat shock(hs) treatment(1). This driver line was mated with a tg(UAS:eGFP-HRASV12)(2) responder line in order to mimic somatic events leading to human oncogene expression. Results:Surprisingly a single heat shock at 30hpf, (referred here as HRASV12hs) induces human HRASV12 expression in hematopoietic progenitors as judged by the expansion of the hematopoietic tissue. Histological analysis showed an increased number of monocytes/macrophages. Blood smear of HRASV12hs larvae showed the expansion of blasts and myeloid precursors. Quantitative analysis of gene expression highlights a remarkable increase of myelo-erythroid restricted genes associated with a slight increase of staminality markers (pu1, mpx, lmo2, mpl, CD41 and c-myb). Moreover fish raised to adulthood developed hyper-plastic kidney marrow, the site of definitive haematopoiesis, the equivalent of mammalian bone marrow. Conclusions and Future Perspectives:The combination of hyper-proliferation of blood cells associated with the expansion of the hematopoietic compartment that we found in this model reproduces the pathological features of human myeloproliferative disorders. This study shows that it is possible to drive the expression of leukemia associated oncogenes to the hematopoietic compartment in developing ZF thus reproducing some of the features of human blood malignancies in an in vivo model. Our Group is now testing different driver lines in order to induce oncogene expression in earliest hematopoietic progenitors and is focusing on different responder lines expressing human oncogenes involved in hematopoietic malignancies. Disclosures:No relevant conflicts of interest to declare.

67* Preclinical evaluation of preventive hypertonic saline therapy for cystic fibrosis-like lung disease in mice

Dehydration of airway surfaces and reduced mucus clearance due to ENaC-mediated increased airway Na+ absorption plays an important role in the pathogenesis of cystic fibrosis (CF) lung disease in patients and causes CF-like lung disease in bENaC-transgenic (bENaC-Tg) mice. Previous clinic trials demonstrated that inhalation of hypertonic saline (HS, NaCl 7%) improved mucus clearance and lung function in CF patients (Donaldson SH et al., 2006), and pilot studies demonstrated that inhalation of HS is well tolerated in young children with CF (Dellon EP et al., 2008). However, effects of HS therapy on airway mucus obstruction and airway inflammation, and the benefits of preventive HS treatment have not been studied. To determine the effects of preventive and late HS therapy on these characteristic features of CF-like lung disease in vivo, we used newborn or 4-week old bENaCTg mice and wild-type littermates and applied intrapulmonary instillation of HS or vehicle alone for 2 weeks. Subsequently, mice were euthanized, bronchoalveolar lavage performed, and lungs processed for histology. We demonstrate that both preventive and late HS treatment provide an effective mucolytic therapy for CF-like lung disease. However, HS promoted airway inflammation in neonates and had no therapeutic effect on airway inflammation in adult bENaC-Tg mice with established CF-like lung disease. These results suggest that potential proinflammatory side effects of early HS treatment should be evaluated before using HS as treatment for CF infants, and that combined mucolytic and anti-inflammatory therapies may be necessary for the treatment of CF lung disease. Supported by EC (MEXT-2004–013666).

Effect of extracellular recombinant human heat shock protein 70 (HSP70) on protein pattern observed after endotoxin-induced macrophage activation

The protein pattern of mouse macrophage strain (J774) has been investigated using 2D electrophoresis after combined action of bacterial endotoxins (LPS), heat shock treatment (HS) and administration of recombinant human Hsp70. The investigation demonstrated significant protective effect of HS and recombinant Hsp70 treatment applied before LPS introduction. This effect is apparently realized by means of several signal transduction systems. In the course of the investigation, we have identified eight proteins, which exhibited pronounced changes in their synthesis due to combined treatment. The data accumulated may shed light on molecular mechanisms underlying protective antiseptic action of HS and/or recombinant Hsp70 applied before LPS administration.

Effects of vagotomy, splanchnic nerve lesion, and fluorocitrate on the transmission of acute hyperosmotic stress signals to the supraoptic nucleus

The response to hyperosmotic stresses in the abdominal cavity is regulated, in part, by vasopressin (VP)-secreting neurons in the supraoptic nucleus (SON). How osmotic stress signals are transmitted to the brain is incompletely understood, and whether the transmission routes for osmotic stress signals differ between acute and chronic stresses is unknown. Here we investigated the role of the vagus, splanchnic nerves, and astrocytes in the SON in transducing acute hyperosmotic-stress signals from the abdominal cavity. We found that acute administration of hyperosmotic saline triggered the activation of neurons as well as astrocytes in the SON and the adjoining ventral glia limitans (SON-VGL). Severing the subdiaphragmatic vagal nerve (SDV) prevented the normal response of cells in the SON to HS treatment and attenuated the release of VP into the bloodstream. Lesioning the splanchnic nerves (SNL) diminished HS-induced release of VP, but to a much lesser extent than SDV. Furthermore, SNL did not significantly affect the up-regulation of Fos in SON neurons or the up-regulation of Fos and GFAP in SON and SON-VGL astrocytes that normally occurred in response to HS and did not affect HS-induced expansion of the SON-VGL. Inhibiting astrocytes with fluorocitrate (FCA) prevented the response of the SON to HS and attenuated the release of VP, similarly to SDV surgery. These results suggest that the vagus is the principle route for the transmission of hyperosmotic signals to the brain and that astrocytes in the SON region are necessary for the activation of SON neurons and the release of VP into the bloodstream.

Altered expression of the natriuretic peptide system in genetically modified heme oxygenase-1 mice treated with high dietary salt

Heme oxygenase-1 (HO-1) has been well established as a cytoprotective molecule, and has been shown to exert cardioprotective effects in both hypertension and cardiac hypertrophy. However, the precise mechanism of the cardioprotective effect of HO-1 has yet to be fully elucidated. With the natriuretic peptide system (NPS) as a key player in cardiovascular homeostasis and tissue dynamics, we sought to examine the effect of high dietary salt treatment in genetic models of HO-1 expression, and assessed the expression of the NPS in the left ventricle (LV), to determine if the effects of altered HO-1 expression may be due to modified levels of the NPS. Age-matched 12-week old male HO-1 knockout (HO-1(-/-)) and HO-1 cardiomyocyte-specific transgenic overexpressing (HO-1(Tg)) mice were treated with either normal salt (NS; 0.8%) or high salt (HS; 8.0%) chow for 5weeks. LV mRNA expression was determined using quantitative real-time PCR. ANP peptide level was measured in the LV and plasma using radioimmunoassay, and LV cyclic 3'-5' guanosine monophosphate level was measured using an enzyme immunoassay kit. HO-1(-/-) fed HS diet had significantly higher left ventricle-to-body weight ratio (LV/BW) compared to HO-1(+/+) mice fed NS diet. HO-1(-/-) mice had significantly reduced expression of the NPS compared to controls, and these mice did not exhibit a salt-induced increase in ANP expression. HS treatment had no noticeable effect on LV/BW in HO-1(Tg) mice compared to controls. HO-1(Tg) mice had significantly higher ANP and BNP expression compared to controls. There were no differences in LV cGMP levels among all genotypes and dietary treatments. HO-1 ablation resulted in significantly lower mRNA expression of the NPS, whereas HO-1 overexpression resulted in higher mRNA expression of the NPS. Both were substantiated by peptide levels as measured by RIA. These data indicate that the detrimental effect of reduced HO-1 expression and the cardioprotective effect of increased HO-1 expression may be due, in part, to altered expression of the NPS.

Contrasting effects of intervention with ETA and ETB receptor antagonists in hypertension induced by angiotensin II and high-salt dietThis article is one of a selection of papers published in the two-part special issue entitled 20 Years of Endothelin Research.

Endothelin (ET) receptor antagonists are antihypertensive and renoprotective in angiotensin II (AngII)-induced hypertension if administered when AngII infusion commences, but their effects on established hypertension are poorly understood. We therefore tested the effects of intervening with an ETA (ABT-627) or ETB (A-192621) receptor antagonist after establishing hypertension with AngII (65 ng/min s.c.) plus 8% NaCl diet (AngII–HS) in rats. Prior to administration of ABT-627, AngII–HS and AngII–HS plus ABT-627 groups displayed robust hypertension (mean arterial pressure (MAP), 170 ± 5 and 165 ± 5 mm Hg versus 110 ± 3 mm Hg in normal salt control rats at day 7, P &lt; 0.05). Administering ABT-627 from day 8 of AngII–HS treatment prevented further rises in MAP (168 ± 5 and 191 ± 3 mm Hg at day 13 in AngII–HS plus ABT-627 and AngII–HS, P &lt; 0.001), without blunting the significant increases in urinary protein (19-fold), albumin (25-fold), or MCP-1 excretion (6- to 8-fold) or the reduction in creatinine clearance. Administering A-192621 from day 8 mildly exacerbated AngII–HS induced hypertension (P &lt; 0.05 for AngII–HS versus AngII–HS plus A-192621 on days 11 and 12 only) and reduced plasma nitrite/nitrate concentration (P &lt; 0.05), without affecting proteinuria, albuminuria, or creatinine clearance. These results confirm the importance of ETA receptor signaling in maintaining AngII–HS hypertension and suggest that including ETB receptor blockade in therapeutic approaches to treating hypertension would be ineffective or even counterproductive.

Enhancing effect of a sea cucumber Stichopus japonicus sulfated polysaccharide on neurosphere formation in vitro

Neural stem/progenitor cells (NSPCs) exhibit therapeutic potential in neuronal diseases. Previously, we reported that a sulfated polysaccharide (HS) from the sea cucumber Stichopus japonicus increased the proliferation of NSPCs. Since the formation of neurospheres is related with NSPCs proliferation, we investigated the mechanism leading to neurosphere formation with and without HS. The results showed that HS significantly promoted neurosphere formation in a dose-dependent manner at concentrations between 2 and 8 μg/ml. Cell cycle analysis showed that HS increased the percentage of cells in S phase by 2.8-fold, as compared with the control. On the other hand, we observed a significantly rapid aggregation of NSPCs, resulting in formation of neurospheres as early as 2 h after HS treatment. However, the aggregation was not caused by chemotactic migration of NSPCs, as evidenced by the transwell chamber assay. Furthermore, the effect of HS on NSPCs was similar to the tumor necrosis factor-α (TNF-α) that activated nuclear factor NF-κB. Thus, we demonstrated that HS was able to promote cell proliferation and aggregation of NSPCs which could lead to the formation of neurospheres, and suggested that HS can serve as an adjuvant for promoting proliferation of NSPCs and formation of neurospheres.

Effect of heat stress and ergopeptine alkaloids on the immune system of rats

Rats were fed diets containing alkaloids (E+), minus alkaloids (E−), or pair‐fed (PF to E+). Groups were further divided into temperature treatments of thermoneutrality (TN; 21°C) or heat stress (HS; 33°C). Rats received diet treatments at TN for 1 week followed by 3 days HS or TN treatment. Feed intake and body weight were reduced by alkaloids, heat stress, and combination of both stressors. Feed intake was reduced 82% from preheat level in E+HS rats compared to 75% in HS rats. Blood samples were collected at trial end and analyzed by flow cytometry. Natural killer (NK) cells increased with all stressors, such that E+HS level was twice that of E‐TN rats (4.2% vs. 2.1%; α=0.05). Caloric restriction (HS and TN) modestly increased NK cells. The combination of stressors dimished B cells by one‐half (α=0.05) compared to E‐TN. Treatment differences in T cell percentage, however, were not statistically significant. In summary, combination of alkaloid intake and heat stress significantly affected the proportion of circulating NK and B cells, with more modest changes in T cells. Caloric restriction (PF) increased NK cells, regardless of environmental conditions. (USDA Agreement No. 58‐6227‐3‐016)

Rumen Fermentation Characteristics and Lactation Performance in Dairy Cows Fed Different Rumen Protected Soybean Meal Products

One hundred lactating Holstein dairy cows were cows were assigned to investigate the effect of untreated Soybean Meal (SBM) by different treated SBM products; heat+ soy hulls addition (HS), extrusion treatment (EP), addition of tannin plant extract and essential oil (PA) or addition of tannin plant + pelleting (HPA) on rumen fermentation, milk production and composition of dairy cows from 17 -25 after calving. th th Basal experimental diet was formulated (containing untreated SBM) and used as control, SBM was replaced by four treated SBM products and fed to the five groups (20 cows per each). Solvent extracted untreated SBM exhibited greater effective degradability of CP and AA when compared with treated SBM products (HS, EP, PA or HPA). This was due to a greater fraction of soluble protein. Moreover, treated SBM products (HS, EP, PA and HPA) contained relatively low concentrations of lysine, arginine, histidine, alanine, praline, serine, aspartic acid and glutamic acid in different levels compared with SE product which suggesting binding and cross linking reactions involving these AA as a result of the treatment methods. Treated SBM feeding instead of untreated one had no effect (p>0.05) on dry matter intake while, improved milk production and milk-to-feed ratio across the whole experimental periods by about (2.2, 1.9, 3.2 and 4.4%) and by (2.5, 1.9, 3.8 and 4.4%) respectively. Moreover, treated SBM reduced (p 0.05) on total VFA, acetate, butyrate concentrations and slightly decreased propionate in the rumen when compared with cows fed on untreated SBM containing diet. Regarding blood serum units treated SBM had no effect (p>0.05) on blood serum glucose concentration, however cows fed on diets containing EP, PA and HPA treated SBM instead of untreated SBM showed a reduction (p 0.05) reduction in blood urea N by about 3.8%. Treated SBM products increased milk fat percentage, fat yield and protein yield and had no effect on milk lactose percentage and the present study suggested that HS and EP treatment methods of SBM is less effective and the cow performance lesser respond than PA or HPA methods which depend on tanninferous plant species that protect protein from degradation in the rumen due to presence of small amounts of condensed tannin in the plant species and may be more available and digestible in the intestine more than the previous processing.

Shade-induced response and recovery of the seagrass Posidonia sinuosa

The effect of shading on the seagrass Posidonia sinuosa Cambridge et Kuo was investigated to identify mechanisms that prolong its survival during periods of low light and permit its subsequent recovery. We also tested whether the responses were consistent in plants growing at different depths. Shade treatments were low (LS; 70 – 100% of ambient Photosynthetic Photon Flux Density), medium (MS; 12 – 39%) and heavy (HS; 5 – 4%) at the shallow (3 – 4 m) site, whilst the deep (7 – 8 m) site had no HS treatment. HS at the shallow and MS at the deep site were below minimum light requirements (MLR) for the long-term survival of P. sinuosa. Physiological, morphological and growth attributes were repeatedly measured during 198 d of shade treatments and a subsequent 384 d recovery period at ambient PPFD. Shoot density declined by 82% within 105 d under HS treatment, though 6% of shoots remained after 198 d. We estimate that complete shoot loss in HS would have taken 2 years. Rhizome sugar concentrations declined to 32 – 52% of the controls at the end of the most severe shading treatments but after shoot loss, sugar concentrations declined more slowly or increased, suggesting a return to positive carbon balance. In the treatments below MLR, shading induced changes in physiological, morphological and growth characteristics, including reduced leaf length and width, reduced δ 13C and photosynthetic adaptation to low light (increased α, reduced E k and ETR max), though not consistently. After removal of shading, photosynthetic characteristics became more typical of high light adaptation, possibly induced by greater light penetration through the thinned canopy, including reversal of the changes in α, E k and ETR max and induction of non-photochemical quenching. Carbohydrate concentrations increased to ambient concentrations within 115 d at ambient PPFD. Recovery of shoot density was slow, remaining significantly reduced in the MS and HS treatments after 384 d recovery. Shoot density at the end of shading is an important determinant of the rate seagrass meadows will recover and we estimated that the moderately and heavily shaded meadows would require 3.5 to 5 years to recover.

Patient-Specific Induced Pluripotent Stem Cells in Hurler Syndrome

Hurler syndrome (HS; mucopolysaccharidosis type I) is caused by severe mutations in the iduronidase (IDUA) gene, leading to multi-organ system dysfunction due to the toxic accumulation of glycosaminoglycans. Although allogeneic hematopoietic cell transplantation (HCT) has been shown to provide the IDUA protein and to reverse many of the manifestations of HS, allogeneic HCT is associated with significant morbidity and mortality. We hypothesized that an advantageous alternative strategy may be to induce gene-corrected autologous pluripotent cells to become hematopoietic stem cells, which then provide the missing IDUA enzyme. Because patient-specific embryonic stem cell isolation is not practical, recent strategies have been developed that reprogram adult cells to acquire pluripotency. Such induced pluripotent stem (iPS) cells can be created from fibroblasts or mesenchymal stromal cells (MSCs). As a first step in testing of iPS cells for gene-corrected HS treatment, we isolated host MSCs from the bone chips of a 9-year-old boy with HS who had undergone spinal surgery 8 years after successful allogeneic HCT. HS-MSCs expressed no IDUA, confirming a lack of contamination from either donor-derived hematopoietic cells or MSCs. To create HS-iPS cells, HS-MSCs were transduced with viral vectors carrying reprogramming transcription factors (OCT4, SOX2, KLF4, and c-MYC) that are typically associated with pluripotency and expressed at high levels in embryonic but not adult stem cells. Transduced cells were cultured on supportive stroma of irradiated mouse embryo fibroblasts. Within several weeks, colonies of iPS cells emerged from the two-dimensional culture. When compared to MSCs, the HS-iPS cells showed persistent mRNA expression of OCT3/4 and SOX2 and transient mRNA expression of c-MYC and KLF4, which is expected to occur in the wild-type iPS cells. HS-iPS cells expressed protein markers characteristic of reprogrammed immature cells: OCT3/4, NANOG, stage-specific embryonic antigens (SSEA) 3 and 4, tumor rejection antigens (TRA) 1–60 and 1–81, and alkaline phosphatase. HS-iPS cells had normal male karyotype as determined by chromosomal G-banding. As a second step in creating gene-corrected HS-iPS cells, we employed the non-viral Sleeping Beauty (SB) transposon system (because of the less random pattern of genome integration when compared to viral vectors). Human HS-iPS cells were co-nucleofected with an SB transposon that harbored the human IDUA gene and an expression cassette of the green fluorescent protein (GFP) along with an SB transposase plasmid that provides the enzymatic machinery necessary for integration into TA dinucleotide sites within the genome. Two weeks after nucleofection 10%-15% of HS-iPS cells expressed GFP. Total glycosaminoglycans (a hallmark of the biochemical defect in HS) in unsorted cultures were decreased to wild-type levels. IDUA expression in unsorted cultures was approximately 10% of wild-type IDUA levels, which is within the range sufficient for phenotypic rescue in HS patients after allogeneic HCT. Experiments are ongoing, and data will be presented in regards to: a) map transposon insertions in the genome to prove stable transgenesis by transposition; b) characterization of the differentiation potential of the corrected HS-iPS cells into various mesodermal lineages relevant to rescue of the clinical phenotype associated with HS (hematopoietic, chondrogenic, and osteogenic); c) assessment of development and consequences of cellular pathology in numerous tissue types affected by IDUA deficiency. To our knowledge these are the first data to report that autologous iPS cells can be obtained from HS patients. In summary, HS-iPS cells present an opportunity to use the hematopoietic progeny of gene-corrected autologous cells clinically in a manner that may preclude the immunologic complications of allogeneic transplantation.

Performance and water-use efficiency (single-leaf vs. whole-canopy) of well-watered and half-stressed split-root Lambrusco grapevines grown in Po Valley (Italy)

‘Lambrusco a foglia frastagliata’ grapevines ( Vitis vinifera L.) were grown outdoors at Piacenza (44°55′N, 9°44′E, Po Valley, Italy) with the root system split between two 30-L pots and subjected from pre-veraison (17 July) to harvest (5 September) to soil drying of half of the root system (HS) induced by withholding water from one of the two pots as compared to well-watered (WW) vines (both pots daily recharged at field capacity). Volumetric soil-water content, pre-dawn and mid-morning leaf water potential, single-leaf gas-exchange as assimilation rate, stomatal conductance and transpiration were monitored throughout the trial. Whole-canopy gas-exchange as net CO 2 exchange rate (NCER) and transpiration were tracked from 31 August to 7 September on three vines per treatment on a 24-h basis using an enclosure method. Primary leaf carbon isotope ( δ 13C) composition, yield components and must composition were determined at harvest. Withdrawing water from one pot triggered a water stress response showing higher stomatal sensitivity to changes in air vapour pressure deficit, relatively low assimilation rates, high intrinsic and extrinsic water-use efficiency (WUE) and earlier cessation of shoot growth. Yet, mid-morning leaf water potential was consistently lower in HS treatment over stress as compared to WW, indicating an anisohydric adjustment. Canopy NCER given on a leaf-area basis showed mean daily rates ranging from 3.9 to 4.9 μmol m 2 s −1 in WW canopies against 2.6–3.0 μmol m −2 s −1 in HS. Conversely, canopy transpiration rates varied from 0.915 to 1.157 mmol m −2 s −1 for WW to 0.630–0.714 mmol m 2 s −1 in HS. Increased leaf-based intrinsic and extrinsic WUE in HS did not match the canopy response, which to some extent resulted in an opposite outcome, i.e. higher canopy WUE in well-watered vines especially in the morning hours. Likewise, δ 13C did not differ between treatments. This suggests caution when point-time determinations of single-leaf-based WUE are extrapolated to the whole-canopy behaviour when assessing the water saving strategies of a given genotype. The stressed vines achieved no variation in yield level and components and had improved grape composition as to soluble solids and total anthocyanins. This optimal behaviour is likely due to earlier shoot growth cessation, enhanced maturity and a buffering leaf-to-fruit ratio (3.61 m 2 kg −1) that mitigated the effects of post-veraison stress.

Effects of thermal and osmotic stress on growth, osmoregulation and Hsp70 in sea cucumber (Apostichopus japonicus Selenka)

This study deals with the effect of thermal and osmotic stress on growth, osmoregulation and Hsp70 level in the sea cucumber Apostichopus japonicus Selenka. Sea cucumbers were exposed to 30 °C (Hs treatment) or 20 °C (control) for 2 h, and then were challenged with osmotic shock, which was administered by transferring the sea cucumbers from natural seawater (about 32 ppt) to different salinities (20 ppt, 25 ppt, 30 ppt, and 40 ppt). After a 30-day experiment, the specific growth rate (SGR) and survival rate of sea cucumbers were measured. To elucidate the underlying physiological mechanisms, osmotic pressure of the coelomic fluid, Na+/K+-ATPase and Hsp70 level of the sea cucumbers from Hs treatment and control were measured at selected times (1 h, 2 h, 6 h, 24 h, and 48 h) after the osmotic shock. At the end of the 30-day experiment, SGR of Hs sea cucumber was higher than that of the control at salinity of 20 ppt. However, SGR of Hs sea cucumber was lower than that of the control at salinity of 30 ppt, 40 ppt and natural seawater. At salinity of 20 ppt, the survival rate of A. japonicus was only 20%. These results indicted that a salinity of 20 ppt exceeded the ecological tolerance for growth in juvenile A. japonicus. A prior heat shock could accelerate growth when the ambient salinity was below the ecological tolerance for growth (salinity of 20 ppt); on the other hand, it could retard growth when the ambient salinity was within the ecological tolerance for growth (salinity of 25 ppt, 30 ppt, 40 ppt and natural seawater). The osmotic pressure of the coelomic fluid changed rapidly and stabilized by 6 h after the osmotic shock. The temporal change pattern of Na+/K+-ATPase activity increased initially and reached the maximum value within 6 h. These results indicated that A. japonicus was an osmotic conformer, and the activity of Na+/K+-ATPase had an adaptive response to the change of ambient salinity. Both thermal and osmotic stresses could induce the up-regulation of Hsp70, which might partly explain the differences in growth of sea cucumbers at different salinities.

Heat shock-mediated H2O2 accumulation and protection against Cd toxicity in rice seedlings

Rice (Oryza sativa L.) seedlings stressed with CdCl2 (0.5 mM or 50 μM) showed typical Cd toxicity (leaf chlorosis, decrease in chlorophyll content, or increase in H2O2 and malondialdehyde contents). Rice seedlings pretreated with heat shock at 45°C (HS) for 2 or 3 h were protected against subsequent Cd stress. Rice seedlings pretreated with HS had similar Cd concentration in leaves caused by CdCl2 as those non-HS. The content of H2O2 increased in leaves 1 h after HS exposure. However, APX and GR activities were higher in HS-treated leaves than their respective control, and it occurred after 2 h of HS treatment. Pretreatment of rice seedlings with H2O2 under non-HS conditions resulted in an increase in APX, GR, and CAT activities and protected rice seedlings from subsequent Cd stress. HS-induced H2O2 production and protection against subsequent Cd stress can be counteracted by imidazole, an inhibitor of NADPH oxidase complex. Results of the present study suggest that early accumulation of H2O2 during HS signals the increase in APX and GR activities, which in turn prevents rice seedlings from Cd-caused oxidative damage.

Measurements of changes in [Ca 2+] i and [Cl −] i during glioblastoma cell movement

The induction of heat shock protein (HSP) 72 by mild electrical stimulation with heat shock (MES + HS), which improves visceral adiposity and insulin resistance in mice, may be beneficial in treating metabolic syndrome (MS) or type 2 diabetes mellitus (T2DM).Using open-label crossover trials, 40 subjects with MS or T2DM were randomly assigned using computer-generated random numbers to 12 weeks of therapeutic MES + HS followed by 12 weeks of no treatment, or vice versa. During the intervention period, physical and biochemical markers were measured.Compared to no treatment, MES + HS treatment was associated with a significant decrease in visceral adiposity (− 7.54 cm2 (− 8.61%), 95% CI − 8.55 to − 6.53 (p = 0.037) in MS, − 19.73 cm2 (− 10.89%), 95% CI − 20.97 to − 18.49 (p = 0.003) in T2DM). Fasting plasma glucose levels were decreased by 3.74 mg/dL (− 5.28%: 95% CI − 4.37 to − 3.09 mg/dL, p = 0.029) in MS and by 14.97 mg/dL (10.40%: 95% CI − 15.79 to 14.15 mg/dL, p < 0.001) in T2DM, and insulin levels were also reduced by 10.39% and 25.93%, respectively. HbA1c levels showed a trend toward reduction (− 0.06%) in MS, and was significantly declined by − 0.43% (95% CI − 0.55 to − 0.31%, p = 0.009) in T2DM. HbA1c level of less than 7.0% was achieved in 52.5% of the MES + HS-treated T2DM patients in contrast to 15% of the non-treated period. Several insulin resistance indices, inflammatory cytokines or adipokines, including C-reactive protein, adiponectin, and tumor necrosis factor-α, were all improved in both groups. In isolated monocytes, HSP72 expression was increased and cytokine expression was reduced following MES + HS treatment. Glucose excursions on meal tolerance test were lower after using MES + HS in T2DM.This combination therapy has beneficial impacts on body composition, metabolic abnormalities, and inflammation in subjects with MS or T2DM. Activation of the heat shock response by MES + HS may provide a novel approach for the treatment of lifestyle-related diseases.Funding for this research was provided by MEXT KAKENHI (Grants-in-Aid for Scientific Research from Ministry of Education, Culture, Sports, Science and Technology, Japan).

Properties of mammalian neural progenitor cells as revealed by genome-wide single cell gene expression profiles

The induction of heat shock protein (HSP) 72 by mild electrical stimulation with heat shock (MES + HS), which improves visceral adiposity and insulin resistance in mice, may be beneficial in treating metabolic syndrome (MS) or type 2 diabetes mellitus (T2DM).Using open-label crossover trials, 40 subjects with MS or T2DM were randomly assigned using computer-generated random numbers to 12 weeks of therapeutic MES + HS followed by 12 weeks of no treatment, or vice versa. During the intervention period, physical and biochemical markers were measured.Compared to no treatment, MES + HS treatment was associated with a significant decrease in visceral adiposity (− 7.54 cm2 (− 8.61%), 95% CI − 8.55 to − 6.53 (p = 0.037) in MS, − 19.73 cm2 (− 10.89%), 95% CI − 20.97 to − 18.49 (p = 0.003) in T2DM). Fasting plasma glucose levels were decreased by 3.74 mg/dL (− 5.28%: 95% CI − 4.37 to − 3.09 mg/dL, p = 0.029) in MS and by 14.97 mg/dL (10.40%: 95% CI − 15.79 to 14.15 mg/dL, p < 0.001) in T2DM, and insulin levels were also reduced by 10.39% and 25.93%, respectively. HbA1c levels showed a trend toward reduction (− 0.06%) in MS, and was significantly declined by − 0.43% (95% CI − 0.55 to − 0.31%, p = 0.009) in T2DM. HbA1c level of less than 7.0% was achieved in 52.5% of the MES + HS-treated T2DM patients in contrast to 15% of the non-treated period. Several insulin resistance indices, inflammatory cytokines or adipokines, including C-reactive protein, adiponectin, and tumor necrosis factor-α, were all improved in both groups. In isolated monocytes, HSP72 expression was increased and cytokine expression was reduced following MES + HS treatment. Glucose excursions on meal tolerance test were lower after using MES + HS in T2DM.This combination therapy has beneficial impacts on body composition, metabolic abnormalities, and inflammation in subjects with MS or T2DM. Activation of the heat shock response by MES + HS may provide a novel approach for the treatment of lifestyle-related diseases.Funding for this research was provided by MEXT KAKENHI (Grants-in-Aid for Scientific Research from Ministry of Education, Culture, Sports, Science and Technology, Japan).

Interleukin-6 and vascular endothelial growth factor release by renal cell carcinoma cells impedes lymphocyte–dendritic cell cross-talk

Anti-tumour T cell response requires antigen presentation via efficient immunological synapse between antigen presenting cells, e.g. dendritic cells (DC), and specific T cells in an adapted Th1 cytokine context. Nine renal cell carcinoma (RCC) primary culture cells were used as sources of tumour antigens which were loaded on DC (DC-Tu) for autologous T cell activation assays. Cytotoxic activity of lymphocytes stimulated with DC-Tu was evaluated against autologous tumour cells. Assays were performed with 75 grays irradiated tumour cells (Tu irr) and with hydrogen peroxide +/- heat shock (Tu H(2)O(2) +/- HS) treated cells. DC-Tu irr failed to enhance cytotoxic activity of autologous lymphocytes in seven of 13 assays. In all these defective assays, irradiated tumour cells displayed high interleukin (IL)-6 and vascular endothelial growth factor (VEGF) release. Conversely, when tumour cells released low IL-6 levels (n = 4), DC-Tu irr efficiently enhanced CTL activity. When assays were performed with the same RCC cells treated with H(2)O(2) + HS, DC-Tu stimulation resulted in improved CTL activity. H(2)O(2) + HS treatment induced post-apoptotic cell necrosis of tumour cells, totally abrogated their cytokine release [IL-6, VEGF, transforming growth factor (TGF)-beta1] and induced HSP70 expression. Taken together, data show that reduction in IL-6 and VEGF release in the environment of the tumour concomitantly to tumour cell HSP expression favours induction of a stronger anti-tumour CTL response.